Rare individuals can naturally clear chronic hepatitis B virus (HBV) infection and acquire protection from reinfection as conferred by vaccination. To examine the protective humoral response against ...HBV, we cloned and characterized human antibodies specific to the viral surface glycoproteins (HBsAg) from memory B cells of HBV vaccinees and controllers. We found that human HBV antibodies are encoded by a diverse set of immunoglobulin genes and recognize various conformational HBsAg epitopes. Strikingly, HBsAg-specific memory B cells from natural controllers mainly produced neutralizing antibodies able to cross-react with several viral genotypes. Furthermore, monotherapy with the potent broadly neutralizing antibody Bc1.187 suppressed viremia in vivo in HBV mouse models and led to post-therapy control of the infection in a fraction of animals. Thus, human neutralizing HBsAg antibodies appear to play a key role in the spontaneous control of HBV and represent promising immunotherapeutic tools for achieving HBV functional cure in chronically infected humans.
Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LPs) at the endoplasmic reticulum (ER) membrane, providing an ...opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV-LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV-B), the virus most closely related to HCV, to identify conserved domains. GBV-B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV-B core is smaller and lacks ~35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133-152 and 153-167 in HCV core) that appear highly conserved in domain II of both GBV-B and HCV core proteins resulted in loss of HCV core ER anchoring and self-assembly into HCV-LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV-B core according to sequence alignment had contrasting effects. Amino acids 15-28 and 60-66 were shown to be dispensable for HCV-LP assembly and morphogenesis, whereas aa 88-106 were required for this process. The production of GBV-B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV-B-LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.
The paramyxo- and pneumovirus family includes a wide range of viruses that can cause respiratory and/or systemic infections in humans and animals. The significant disease burden of these viruses is ...further exacerbated by the limited therapeutics that are currently available. Host cellular proteins that can antagonize or limit virus replication are therefore a promising area of research to identify candidate molecules with the potential for host-targeted therapies. Host proteins known as host cell restriction factors are constitutively expressed and/or induced in response to virus infection and include proteins from interferon-stimulated genes (ISGs). Many ISG proteins have been identified but relatively few have been characterized in detail and most studies have focused on studying their antiviral activities against particular viruses, such as influenza A viruses and human immunodeficiency virus (HIV)-1. This review summarizes current literature regarding host cell restriction factors against paramyxo- and pneumoviruses, on which there is more limited data. Alongside discussion of known restriction factors, this review also considers viral countermeasures in overcoming host restriction, the strengths and limitations in different experimental approaches in studies reported to date, and the challenges in reconciling differences between in vitro and in vivo data. Furthermore, this review provides an outlook regarding the landscape of emerging technologies and tools available to study host cell restriction factors, as well as the suitability of these proteins as targets for broad-spectrum antiviral therapeutics.
Fibrinogen‐β (FBG‐β), an important acute‐phase protein (APP), is generated by the liver as a target for inflammatory mediators. Here we identified FBG‐β as a hepatitis C virus (HCV) core interacting ...protein by screening a human liver complementary DNA (cDNA) library using mammalian two‐hybrid analysis. An association between FBG‐β and HCV core protein was verified by confocal microscopy and coimmunoprecipitation from the transfected human hepatocyte (Huh‐7) cell line. HCV core or genomic RNA transfected Huh‐7 cells modestly increased FBG‐β protein expression when compared to the basal level in control hepatocytes. Transfection of HCV core or full‐length (FL) gene into Huh‐7 cells up‐regulated basal FBG‐β promoter activity. Exogenous addition of IL‐6 stimulates FBG‐β promoter activity in hepatocytes. However, ectopic expression of HCV core or FL in hepatocytes inhibited IL‐6‐stimulated FBG‐β promoter activation. Inhibition of endogenous FBG‐β expression following introduction of small interfering RNA (siRNA) into cells displayed a gain of function of promoter regulation by HCV core protein. Further studies suggested that HCV core gene expression in stable transfectants of Huh‐7 cells resulted in a basal up‐regulation of FBG‐β and other APPs. However, treatment with cytokines, interleukin‐6 (IL‐6), or tumor necrosis factor‐α repressed FBG‐β and other acute‐phase response (APR) genes. Conclusion: Our results reveal that the core/FBG‐β interaction may act as a regulatory feedback, allowing repression of IL‐6‐stimulated APR genes. Together, these data suggested a network of interactions between HCV core and the hepatic APR genes, and may contribute to impaired innate immunity for viral persistence. (HEPATOLOGY 2010.)
TLR7 agonists are of high interest for the treatment of cancer, auto-immunity and chronic viral infections. They are known to activate plasmacytoid dendritic cells (pDCs) to produce high amounts of ...Type I Interferon (IFN) and to facilitate T and B cell responses, the latter with the help of maturation markers such as CD40, CD80 and CD86. The TLR7 single nucleotide polymorphism (SNP) rs179008 (GLn11Leu), sex and chronic viral infection have all been reported to influence pDC IFN production. It is unknown, however, whether these factors also influence pDC phenotypic maturation and thereby IFN-independent pDC functions. Furthermore, it is unclear whether SNP rs179008 influences HBV susceptibility and/or clearance.
Here we investigated whether the SNP rs179008, sex and HBV infection affected phenotypic maturation of pDCs from 38 healthy individuals and 28 chronic HBV patients. In addition, we assessed SNP prevalence in a large cohort of healthy individuals (n = 231) and chronic HBV patients (n = 1054).
Consistent with previous reports, the rs179008 variant allele was largely absent in Asians and more prevalent in Caucasians. Among Caucasians, the SNP was equally prevalent in healthy and chronically infected males. The SNP was, however, significantly more prevalent in healthy females than in those with chronic HBV infection (42 versus 28%), suggesting that in females it may offer protection from chronic infection. Ex vivo experiments demonstrated that induction of the co-stimulatory molecules CD40 and CD86 by TLR7 ligands, but not TLR9 ligands, was augmented in pDCs from healthy SNP-carrying females. Furthermore, CD80 and CD86 upregulation was more pronounced in females independent of the SNP. Lastly, our data suggested that chronic HBV infection impairs pDC maturation. These findings provide insight into factors determining TLR7 responses, which is important for further clinical development of TLR7-based therapies.
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•The TLR7 SNP rs179008 variant is more prevalent in healthy than in chronic HBV-infected females.•TRL7 SNP rs179008 and sex both affect pDC phenotypic maturation in response to TLR7 ligation.•Chronic HBV infection suppresses upregulation of CD40 on pDC upon TLR7 ligation.•TLR7 SNP rs179008 genotype, sex and HBV infection status may influence the response to TLR7 targeted Immunotherapy.
Hepatitis B virus (HBV) is a major cause of acute and chronic hepatitis in humans. As HBV itself is currently viewed as a non-cytopathic virus, the liver pathology associated with hepatitis B is ...mainly thought to be due to immune responses directed against HBV antigens. The outcome of HBV infection is the result of complex interactions between replicating HBV and the immune system. While the role of the adaptive immune response in the resolution of HBV infection is well understood, the contribution of innate immune mechanisms remains to be clearly defined. The innate immune system represents the first line of defense against viral infection, but its role has been difficult to analyze in humans due to late diagnosis of HBV infection. In this review, we discuss recent advances in the field of innate immunity to HBV infection.
Host cell restriction factors are intracellular proteins that can inhibit virus replication. Characterisation of novel host cell restriction factors can provide potential targets for host-directed ...therapies. In this study, we aimed to assess a member of the Tripartite-motif family protein (TRIM) family, TRIM16, as a putative host cell restriction factor. To this end, we utilized constitutive or doxycycline-inducible systems to overexpress TRIM16 in HEK293T epithelial cells and then tested for its ability to inhibit growth by a range of RNA and DNA viruses. In HEK293T cells, overexpression of TRIM16 resulted in potent inhibition of multiple viruses, however, when TRIM16 was overexpressed in other epithelial cell lines (A549, Hela, or Hep2), virus inhibition was not observed. When investigating the antiviral activity of endogenous TRIM16, we report that siRNA-mediated knockdown of TRIM16 in A549 cells also modulated the mRNA expression of other TRIM proteins, complicating the interpretation of results using this method. Therefore, we used CRISPR/Cas9 editing to knockout TRIM16 in A549 cells and demonstrate that endogenous TRIM16 did not mediate antiviral activity against the viruses tested. Thus, while initial overexpression in HEK293T cells suggested that TRIM16 was a host cell restriction factor, alternative approaches did not validate these findings. These studies highlight the importance of multiple complementary experimental approaches, including overexpression analysis in multiple cell lines and investigation of the endogenous protein, when defining host cell restriction factors with novel antiviral activity.
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